In recent years it has become possible to modulate the immune system to improve its response and, where components of the system are non-functioning, to either partially or completely restore the function of the component. For example, bone marrow transplantation is used to replace stem cells or provide missing stem cells to cure severe combined immunodeficiency. In another example, immune cells are removed from cancer patients, treated, and returned to the patient wherein there is tumor regression. (Hwu and Rosenberg, 1994a; Hwu and Rosenberg, 1994b) Further, components of the humeral immune system such as .gamma.-globulin and intravenous immunoglobulin (IVIG) are finding wide therapeutic applications (DeSimone et al., 1990; Hall, 1993). Other immune system components and agents are also being used as therapeutics. (Hadden & Smith, 1992; Hadden, 1993).
Immunomodulators are compounds that modify immune function or have a positive or negative effect on the activity of the immune system. The use of immunomodulators in clinical medicine includes the reconstitution of immune function (or the correction of immunodeficiency) and the suppression of normal or excessive immune function. A major class of immunomodulators is cytokines. Through recombinant technology, many of the cytokines are now available. However, the immune system is complex and the interaction of various components is often necessary to effectively modify immune functions. It would be useful to design preparations that provide the various components and interactions to effectively regulate immune function.
Cytokines are peptide/protein immunomodulators that are produced by activated immune cells including thymus-derived T lymphocytes (T-cells), B lymphocytes and monocyte/macrophages. The cytokines include interleukins (IL-1 through IL-15), colony stimulating factors (CSFs) for granulocytes and/or macrophages (CSF-G, CSF-M, CSF-GM), tumor necrosis factors (TNFs .alpha. & .beta.), and interferons (IFN .alpha., .beta. & .gamma.).
Interleukin-2 (IL-2) is a lymphokine initially described as a T-cell growth factor (Morgan et al., 1976) IL-2 induces and supports proliferation of antigen or mitogen stimulated T-cells. In addition to the T lymphocyte stimulating function, IL-2 is important in such processes as the initiation, expansion and regulation of the immune response, the production of gamma-interferon (IFN.gamma.), the induction of lymphokine-activated killer (LAK) cells, the propagation of cytolytic T-cells, and the augmentation of the killer activity of natural killer (NK) cells. Recombinant IL-2 (rIL-2) is a non-glycosylated protein that is produced by the human cDNA sequence (Taniguchi et al., 1983; Devos, 1983; U.S. Pat. Nos. 4,604,327, 4,569,790 and 4,518,584)
Various individual cytokines, both natural and recombinant, have been investigated for the treatment of cancer and other diseases. For example, recombinant interferon .alpha..sub.2 (rIFN .alpha..sub.2) is approved by the U.S. Food & Drug Administration (FDA) for treatment of Hairy cell leukemia, Kaposi's sarcoma, condyloma accumenata, and chronic hepatitis. Natural IFN.alpha.s, as a mixture (Alferon.RTM.) of the twenty or more made by leukocytes, is licensed for condyloma accumenata. Recombinant IFN-.gamma.(rIFN-.gamma.) is licensed for chronic granulomatous disease. rIL-2 is licensed for renal cell cancer. These and other rIL's and rIFNs are under active evaluation in a variety of diseases including several forms of cancer.
Further, rIL-2 cancer therapy has been explored in many clinics and research centers. Rosenberg and colleagues (Rosenberg et al. 1985, 1987; Mul e and Rosenberg, 1987; Chang and Rosenberg, 1989; Belldegrun and Rosenberg, 1989 and Rosenberg, 1994) have reported the use of systemically administered rIL-2 in the immunotherapy of patients with renal cell cancer, pulmonary cancer and melanoma. Cortesina et al. (1988, 1994) described the effects of loco-regional injections of natural and rIL-2 in head and neck cancer patients and found natural IL-2 to be more effective in yielding tumor regression. Also, patients given large doses of rIL-2 have suffered life threatening toxicity. (Rosenberg et al., 1987).
The development and commercial availability of genetically (recombinant) engineered immunomodulators has accelerated the evaluation of these agents in the cancer clinic. The limited efficacy and significant toxicity associated with high doses of rIL-2, rIFN-.gamma., rTNF-.alpha., and other monotherapies, suggests reconsideration of natural combinations of cytokines in therapeutic strategies. Furthermore, more than one-hundred different cytokine activities have been identified, which raises significant doubt as to whether immunotherapy, based upon combining recombinant cytokines, has a reasonable probability of success in the cancer clinic in the near future.
For example, while IL-2 can stimulate T lymphocyte proliferation as a T-cell growth factor, a number of other factors including other interleukins and thymic peptides are produced in the thymus and are also considered necessary for T lymphocyte development and function. (Hadden, 1992).
An uncharacterized natural interleukin preparation (NI) has been shown by applicants to be effective in promoting T lymphocyte development. This uncharacterized mixed preparation (also referred to as buffy coat interleukin, BC-IL) stimulated the proliferation of prothymocytes, immature and mature thymocytes in vitro more effectively than an equivalent concentration of rIL-2 (Hadden et al., 1989). The NI preparation augmented T lymphocyte development in neonatal mice while rIL-2 was inactive (Hadden et al., 1989) and augmented T lymphocyte development and function in hydrocortisone-treated, aged mice while rIL-2 in equivalent dose was inactive (Hadden et al., 1992b). The NI preparation in low dose prolonged the life in mice bearing malignant melanoma; rIL-2 in equivalent dose was inactive (Kameda et al., 1992). These findings indicated that natural interleukins mixtures have activities not provided by IL-2.
Attempts to correct T lymphocyte defects have been tried experimentally in a variety of settings including T lymphocyte depletion (lymphocytopenia) and T lymphocyte dysfunction (anergy) occurring in aging, cancer, AIDS, and other immunodeficiencies. For example, rIL-2 and thymic peptides have been used in AIDS (HIV) virus infection with variable results (Hadden, 1991). High dose rIL-2 by continuous infusion has been shown to transiently increase T lymphocyte counts in blood of patients with HIV infection but with considerable toxicity (Lane and Fauci, 1986). Pegylated rIL-2 at one and three million units yielded less toxicity but only minor effects on lymphocyte counts in humans with HIV infection (Merigan, 1993). An NI preparation significantly augmented T lymphocyte counts in lymphocytopenia cancer patients without toxicity (Hadden et al, 1994). These findings indicate that natural interleukins act in humans in low doses to increase T cells without toxicity and that rIL-2 while active at high doses is too toxic for medical use. These findings also support the extrapolation of murine data to man.
The above indicates that the use of preparations of naturally occurring cytokines combined with other factors may be more efficient in affecting the immune system with less toxicity. However, the preparations that are currently available are not well characterized and are cumbersome to produce. In order to reproducibly modulate the immune system it would be useful to have well characterized preparations of cytokines that can be produced easily and from which it will be possible to establish reproducible low-toxicity dosages. Prior work by the applicant (Hadden et al., 1992a) and the co-pending patent application U.S. Ser. No. 08/610,075 by the same applicant and assigned to the assignee of the present application and incorporated herein by reference provides such natural cytokine preparations (NI, NIM or NCM).
Deficiencies of cellular immunity in man have also been treated with various thymic hormone/peptide preparations e.g. thymostimulin, Thymosin fraction IV, Thymosin .alpha..sub.1, zinc-thymulin, thymopoietin, thymopentin, and thymic humoral factor (Shuloff, 1985). Several of these preparations are licensed for clinical use in European countries, especially Italy and Germany.
Thymosin .alpha..sub.1 (T-.alpha.1) is a 28 amino acid peptide initially extracted from bovine thymus and later synthesized. (Goldstein et al., 1977; U.S. Pat. Nos. 4,079,127, 4,148,788, 4,293,455, 4,504,415) Thymosin .alpha..sub.1 has been used experimentally to treat cellular immune deficiency and cancer in mice and humans (Goldstein, 1993). It is licensed in Italy for use with influenza vaccine to improve immunization, and it is in trials in chronic hepatitis and breast and lung cancer with encouraging results.
Based upon its immunopharmacology, Thymosin .alpha..sub.1 promotes T lymphocyte function, but none of the defined thymic peptides including Thymosin .alpha..sub.1 have been shown unequivocally to reverse thymic involution and to increase T lymphocyte number.
Analogs and fragments of Thymosin .alpha..sub.1 have been shown to have an effect upon the immune system as set forth in U.S. Pat. Nos. 4,116,951, 4,353,821, 4,466,918, 4,470,926, 4,612,365, 4,910,296. Prothymosin and Thymosin .alpha..sub.11 also mimic some of the actions of Thymosin .alpha..sub.1 and may induce its production by macrophages (Maric et al., 1991; Frillingos et al., 1992; U.S. Pat. Nos. 4,659,694, 4,716,148 and 4,614,731).
Applicants have shown that Thymosin fraction V, a crude thymic extract containing in excess of 35 peptides, has no effect by itself on thymic weight, lymphocyte content, or function in hydrocortisone-treated, aged mice. However, Thymosin fraction V augmented the effect of a natural interleukin preparation on responses of splenocytes and thymocytes to mitogens and interleukins (Hadden et al., 1992b).
It would therefore be useful to combine a natural cytokine preparation with thymic peptides, such as Thymosin .alpha..sub.1, so that the combination can be used therapeutically in the treatment of diseases and other conditions which include reduced function, development and number of T lymphocytes, i.e. cellular immune deficiency.